Exploring the Antidiarrheal Properties of Papaya Leaf: Insights In Vivo Study in Mice-Model and In Silico Analysis at M3 Muscarinic Acetylcholine Receptor Interaction

Diarrhea caused by gut motility involves 5-HT3 serotonin receptor (5-HT3R) antagonist, M3 muscarinic acetylcholine receptor (M3-AchR), and H1 histamine receptor (H1R) via their respective agonist. Papaya (Carica papaya L.) leaf is an herbal medicine to treat diarrhea in Indonesia, though this has not yet been proven scientifically. This study aimed to determine the antidiarrheal properties of papaya leaf through in vivo and in silico studies. In the mice model, papaya leaves were infused with distilled water and subjected to in vivo antidiarrheal study using castor oil-induced diarrhea. In silico molecular docking study of nineteen secondary metabolites was carried out on the M3-AchR (PDB ID: 5ZHP) using AutoDock Tools 1.5.6, while ADMET was predicted by pre-ADMET. The results showed that papaya leaf infusion caused a decrease in the total number of feces, an increase in the onset time of diarrhea, a reduction in the frequency of diarrhea, and an increase in the percentage of inhibition of diarrhea. Quercetin 3-rutinoside, a flavonoid glycoside, is potentially an antidiarrheal remedy at M3-AchR. ADMET prediction showed good distribution on the target and was not toxic, but absorption needed to be considered. We concluded that the antidiarrheal activity of papaya leaf infusion was dose-dependent. Based on a molecular docking study, the flavonoid glycoside was very effective as an antidiarrheal remedy. ADMET prediction showed a specific distribution to the target and was not toxic.

M-AchR, namely, M3, plays an important role in various diseases, such as irritable bowel syndrome (IBS) [5,6].Excess ACh within the nervous system controls smooth muscle, cardiac muscle, and endocrine-exocrine functions leading to diarrhea (D-IBS) [5].Te pathophysiology of D-IBS occurs in M 3 -AchR, so it becomes a specifc treatment target [3].Terefore, drugs that block these receptors, such as anticholinergics (loperamide, loratadine, baclofen, amitriptyline, oxybutynin, and chlorphenamine) are needed, which can be used to reduce motility and GI secretion, thereby alleviating the symptoms of diarrhea [7].However, these drugs have side efects, such as headaches, memory problems, decreased cognitive function, behavioral disorders, anxiety, and insomnia [8].
Te World Health Organization (WHO) supports eforts to improve the safety and efcacy of herbal medicines.In addition, it is recommended that herbal medicine is used in maintaining public health and preventing and treating diseases because of the fewer side efects.About 80% of the world's population in developing countries, including Indonesia, use herbal medicine for their primary health [9].So far, only guava (Psidium guajava L.) leaves are available in the Indonesian market.So, it needs to be developed using other herbal ingredient medicines with the same potential as antidiarrhea, such as papaya (C.papaya L.) leaves.
Papaya, a fowering plant in the Caricaceae family, is native to tropical America, i.e., Southern Mexico and neighboring Central America.It has spread to tropical countries, including Indonesia [10,11].In Indonesia, the area and production of papaya reached 107,813 hectares, with a total production of 1,089,578 tons in 2022 [12].Papaya leaves have been used for lactation, medicine for cancer, digestion, and dengue fever [13].It also has an activity of antiinfammatory, antidiabetic, immunomodulatory, antiviral, increased platelet [14], antimalarial, and antiplasmodial [15].Papaya leaves showed no harmful efects on long-term administration [16].Phytochemical screening showed that papaya leaves contain saponins, cardiac glycosides, anthraquinones, reducing sugars, favonoids, alkaloids, and tannins [17].Even though papaya has several critical medicinal properties, studies have yet to be performed to investigate its antidiarrheal properties.Terefore, this study used an experimental (animal model) approach to examine the antidiarrheal activity of secondary metabolites in the infusion of papaya leaves and a computational in silico molecular docking and ADMET prediction for the frst time.

Infusion Preparation and Phytochemical
Screening.4, 8, and 16 g of papaya leaves were extracted with 100 mL of distilled water for 15 min at 90 °C.Infusion was fltered to 100 mL of volumetric fask and diluted to volume with distilled water [18].Te infusion concentration was 40, 80, and 160 mg/mL, respectively.Phytochemical screening was conducted with methods described by Tiwari et al. [19] and Benne et al. [20] to identify polyphenols, favonoids, tannins, alkaloids, saponins, monoterpenoids, terpenoids, and steroids.
Te infusion reacted with (a) sodium hydroxide solution will develop a dark yellow solution, then become colorless if diluted acid is added, and (b) lead acetate solution will develop yellow precipitate.Te color change indicates favonoids presence.Te infusion is treated with ferric chloride solution and will develop a bluish-black solution to indicate phenols' presence.Te infusion added to a 1% gelatin solution containing sodium chloride will develop a white precipitate to indicate tannins' presence.Te infusion is shaken for 15 min to develop (a) a foam layer 1 cm thick and (b) a foam lasting for 10 min.Te foams indicate saponins' presence [19,20].
Te infusion is dissolved in dilute hydrochloric acid and fltered.(a) Te fltrate is reacted with Mayer's reagent to develop a yellow precipitate.(b) Te fltrate is made to react with Wagner's reagent to develop a brown or reddish precipitate.(c) Te fltrate is reacted with Dragendrof's reagent to develop a red precipitate.Te various precipitates indicate alkaloids' presence [19,20].
Te infusion is evaporated until dry, and then extracted with chloroform.We add 5 drops of acetic anhydride followed by sulfuric acid from the side of the test tube to develop a blue ring at the junction of the two fuids indicating steroids' presence.Te infusion added to 2 mL of chloroform, shaken, and fltered.We add 5 drops of concentrated sulfuric acid to the fltrate and shake to develop a yellow precipitate, which indicate triterpenoids' presence [20].

Antidiarrheal Activity Assay against Castor Oil-Induced
Diarrhea in Mice.Mice fasted for 24 hours and were randomly divided into fve groups of fve mice in each group (n � 5).Group I was administered 0.5 mL of 1% PGA suspension orally and served as a negative control (NC).Group II was administered 0.5 mL of loperamide HCl (5.2 μg/mL) orally and served as a positive control (PC).Groups III, IV, and V were administered an oral dose of 0.5 mL of papaya leaf infusion at 40, 80, and 160 mg/mL, respectively.One 2 Scientifca hour after administering the test doses, the mice received 0.5 mL castor oil orally to induce diarrhea, then individually placed in cages with the foor lined with flter paper to observe the total number of feces, onset time of diarrhea, and frequency.Te total feces were weighed every 30 min for 3 hours, and the papers were changed every hour after each evaluation.Te mean number of control group feces was considered to be 100%.Te percentage inhibition of diarrhea (%) for all the groups was calculated compared to the negative control by using the following equation: inhibition (%) � ((TD control -TD test group)/TD control) × 100, where TD control � total feces of the negative control group and TD test group � total feces of the test or positive control group [21].

Selection of Compounds for Molecular Docking Study.
Te native ligand was separated from water molecules and protein using Discovery Studio 2016 Client ® (DS) (BIOVIA, San Diego, CA, USA) [22] by the script menu, and the selected water molecules and protein chains were saved [23].

Molecular Docking and ADMET Prediction for Antidiarrheal Activity.
Te three-dimensional X-ray crystal structure of the protein used for molecular docking was obtained from the Protein Data Bank (https://www.rcsb.org/),i.e., M 3 -AchR (PDB ID: 5ZHP).Molecular docking was conducted with DS and ADT.Protein was separated from native ligands using DS [22].Ten, it was prepared again with ADT by adding the Kollman charge and hydrogen polar [23].Parameter setting of the grid and docking was carried out in ADT.Determination of the grid box on the active site in the form of the box parameter location and the grid box size (distance, Å) using the ADT [27].Te result obtained was the coordinate of the central grid point (x, y, and z) used as a reference for docking papaya leaf's phytochemical constituents to the M 3 -AchR.For docking parameters based on the Lamarckian genetic algorithm (100 times) with a value of 27,000 (algorithm generation), 2,500,000 (energy evaluation), as well as 150 (population) [28].
Te software parameter was validated with the root mean standard deviation (RMSD) value of the result of redocking as close as the crystallographic value in the selected active site area [29].Te molecular docking process for the compounds of papaya leaves was carried out the same way as the validation process, which uses grid parameters and the docking of the validation result.Te results were observed in the form of binding afnity, including free energy of binding (ΔG) and inhibition constant (Ki), as well as amino acid residues [30].Te output of molecular docking was a notepad fle, and then the lowest bond energy value (best pose) was selected based on the cluster rank.Visualization of the position and orientation of compounds when interacting with protein, the amino acids, and bonds formed using the DS [22].At the same time, pharmacokinetic properties from constituents of papaya leaves were predicted by pre-ADMET (https://preadmet.bmdrc.kr/).

Statistical Analysis.
Results were presented as mean-± standard error of the mean.All comparisons were made by using one-way ANOVA followed by Dunnett's test.A p value of less than 0.05 was considered statistically signifcant.

Infusion Preparation and Phytochemical Screening.
A total of 47.38 g of papaya leaves produced 15.44 g of dried leaves, so the yield was 32.59%.Te percentage yield shows that the papaya leaves had less water content than others.Te infusion of papaya leaves was light green with a characteristic papaya odor.Phytochemical screening based on color change or precipitation showed that papaya leaves contain polyphenols, tannins, favonoids, alkaloids, and saponins.

Molecular Docking Study for Antidiarrheal Activity.
Te validation method for the software was declared valid and is presented in Table 1.Tis is because the redocking ligand value (0.996 Å) was smaller than the crystallographic value (3.10 Å).Visualization of native ligand cocrystal by redocking is shown in Figure 2.
Table 2 of the molecular docking results shows that the favonoid from papaya leaves had a good docking score compared to other compounds.Te docking results of triplicates can be seen in the supplementary data (available here).It is based on the binding afnity value, where quercetin 3-rutinoside was − 11.50 kcal/mol and 3.75 nM Scientifca against the M 3 -AchR.Although quercetin 3-rutinoside, a favonoid containing glycosides, has a binding afnity value that is slightly diferent from the standard drug, it is not so signifcant that it has potential as an antidiarrheal remedy.Te group of alkaloids (carpaine and dehydrocarpaine II) has no interaction with M3-AchR because of the value of ΔG > 0. It showed that not all alkaloid secondary metabolites from papaya leaves have a binding afnity to the active sites of the receptor [31].
A study of the docking fts of each compound has different interactions when it binds to the target protein or enzyme.Table 2 shows the hydrogen bonds between compounds with M 3 -AchR.Te compounds formed at least three hydrogen bonds (conventional, carbon, and pi-donor hydrogen bonds) with various amino acid residues, except for the alkaloid group, which tends to form Van der Waals bonds.Molecular docking for favonoids and favonoid glucosides is shown in Figure 3 for 2D and Figure 4 for 3D.    2 is the average value of three repetitions.

Scientifca
Pharmacokinetic properties include absorption (human intestinal absorption (HIA) and human colon adenocarcinoma cells (Caco-2)), distribution (plasma protein binding (PPB) and blood-brain barrier (BBB)), metabolism (CYP2D6 and CYP3A4 as substrate and inhibition), and toxicity test (mutagenic and carcinogenic) were predicted in compounds of papaya leaves before further stages (clinical trials).Table 3 shows that kaempferol and loperamide have an absorption value between 70-100%, which showed promising results when in the intestine.At the same time, the other compounds were moderately and poorly absorbed.
Te infusion was chosen as the extraction method in this study because it adjusts the empirical use of papaya leaves in Indonesian society, which are boiled for 15 min using water.
It causes a high concentration for the in vivo test.Te results of the phytochemical screening of papaya leaves from Bandung were the same as those of Patil et al. [17].Terefore, the secondary metabolites for molecular docking and ADMET prediction were taken from the secondary metabolites that have been characterized.Papaya leaves contain various phenolic compounds, such as kaempferol, protocatechuic acid, quercetin, 5,7-dimethoxy coumarin, cafeic acid, p-coumarin acid, and chlorogenic acid.Tese  Scientifca compounds are also the major isolates in the leaves, i.e., quercetin 3-O-α-1C4-rhamnopyranoside, quercetin-3-Oglucopyranoside, and quercetin-3-O-rutinoside [34,35].
Castor oil contains ricinoleic acid, which irritates the intestinal mucosa, causing infammation and release of prostaglandins, stimulating gastrointestinal secretion, motility, epithelial permeability, and edema of the intestinal mucosa.Tis prevents sodium, chloride, and water reabsorption, thus causing diarrhea [36].Loperamide HCl is an antidiarrheal standard due to antagonist diarrhea induced by castor oil due to antisecretory and antimotility activities.It blocks M 3 -AchR, causing muscle relaxation [37].Papaya leaf infusion exhibited signifcant antidiarrheal activity in suppressing the propulsive movement of gastrointestinal transit, which indicates the capability to reduce the frequency of feces in diarrhea conditions [38].Te same study was conducted by Terefe et al., 2023, on animals screened to respond to castor oil by inducing watery diarrhea with an efective 400 mg/kg dose.It is because castor oil induces diarrhea by releasing nitrate oxidation, thereby increasing the permeability of the GI membrane to calcium, stimulating prostaglandin synthesis, and causing increased fuid and electrolyte entry into the bowel lumen and peristalsis [39].
Acetylcholine acts on M3 muscarinic receptors to initiate intestinal motility [40].Antidiarrheal activity is predicted to be caused by the relaxant activity of antagonizing muscarinic acetylcholine receptors.Medicinal plants containing secondary metabolites with complementary pharmacology activity and synergism mechanisms are predicted to treat various diseases [41].Several parts of plants, extracts, and plant-derived products have been widely used as medicine [42].Plants are generally rich in secondary metabolites with various biological activities, which naturally act as a defensive mechanism against bacteria, insects, viruses, and fungi [41].
Te in vivo results showed a signifcant diference between the negative control, test, and positive groups, i.e., loperamide HCl. Figure 1 shows that the increased infusion dose was proportional to the increased antidiarrheal activity.Tese were observed from a decrease in the total number of feces, an increase in the onset time of diarrhea, a reduction in the frequency of diarrhea, and an increase in the percentage of inhibition of diarrhea.It concluded that the antidiarrheal activity of papaya leaf infusion was dose-dependent.
Te aqueous papaya leaves extract good antidiarrheal activity and are safe at 200 mg/kg in the rats' model [32].Tis study showed the best antidiarrheal activity by infusion of 4000 mg/kg, equal to loperamide HCl as the standard.Te infusion dose was observed to be 20 times higher than the extract dose.However, if converted from the yield of 8.78% of the extract, 200 mg of the extract would be equivalent to 2.28 g of leaves [32], while 4000 mg infusion was equivalent to 16 g of leaves.So, the infusion dose was only 7.01 times higher than the extract.It was due to a diferent extraction method, i.e., maceration was conducted for 3 days using methanol [32], while infusion was performed for 15 min using distilled water.Te dose diference showed the different concentrations of the extracted secondary metabolites.Applying papaya leaf infusion has two advantages, i.e., the content of secondary metabolites that have antidiarrheal activity and water to prevent dehydration.Tis fnding may support the acclaimed efect of papaya leaves as an antidiarrheal agent in traditional remedies to develop into herbal medicine.Herbal medicine has a more signifcant role in developing countries in overcoming drug resistance, toxicity, adverse efects, and increasing costs if synthetic products are used [47].
Papaya fruit has been proven to have antidiarrheal activity [32].Our study is the frst to determine papaya leaf's antidiarrheal activity in a mice model induced by castor oil.Tis study was conducted based on the results of our phytochemical screening, which showed the presence of polyphenols, tannins, favonoids, alkaloids, and saponins in papaya leaves.Te secondary metabolite content of papaya leaf is similar to papaya fruit, namely, carbohydrates, tannins, saponins, proteins and amino acids, alkaloids, phenolic compounds, and phytosterols [32].We chose papaya leaves over fruit because papaya leaves are always available, while papaya fruit is available, and you have to wait to fower until it fnally produces fruit.Tis is the novelty and advantage of our study if the papaya leaf is developed into a candidate for herbal antidiarrheal preparations because around 80% of the world's people, including Indonesian, have used herbal plants as a substitute for chemical drugs, mainly for reasons of side efects and expenses [48].Antidiarrheal activity of medicinal plants is caused by tannins, favonoids, alkaloids, saponins, reducing sugars, sterols, and glycosides [49,50].Te antidiarrheal activity of papaya leaf infusion is attributed to the presence of secondary metabolites.Papaya leaf infusion contains polyphenols, tannins, favonoids, alkaloids, and saponins.Flavonoids inhibit the release of autacoids and prostaglandins [51,52].Tannins make intestinal mucosa more resistant, reduce secretion, and stimulate normalization of water transport across the mucosal cells, and reduce intestinal transit [53].Saponins inhibit histamine release [52].Flavonoids, tannins, and phenolic compounds also form complex cell walls, binding to adhesion, thus providing antimicrobial activity [50,53].Tese provide a scientifc fnding for the potential of papaya leaves in antidiarrhea.Papaya leaf infusion suppressed the movement of gastrointestinal transit, which is shown from the onset time and frequency of feces in diarrheal conditions.
Computational studies were efectively used to predict the binding afnity of target ligands and very well demonstrated possible molecular mechanisms for pharmacological response [54].Binding afnity is an important factor that needs to be considered when ligand-receptor interactions occur.If the binding afnity is low, the ligand (compound) requires less energy to bind to the receptor.So, low binding afnity values have more signifcant potential to 8 Scientifca interact with target macromolecules [55].Free energy of binding analysis aims to determine the spontaneity of a reaction and the stability of the interaction between the ligand receptors indicated by negative ΔG.In addition, the stability of the interaction is proportional to the compound's potential when it forms strong chemical bonds [56].Te smaller the number or the negative of ΔG indicates a lot of energy to form a stronger bond [57].Experimentally, ΔG was closely related to Ki.According to the equation of ΔG � − RT ln Ki, the value of ΔG was used to predict the ability of compounds to inhibit macromolecules [58].In the present study, nineteen secondary metabolites in three major compounds from the papaya leaves, i.e., alkaloids, favonoids and favonoid glycosides, as well as phenol and phenolic acid, were investigated against M 3 -AchR and the docking scores obtained for all compounds have been reported in Table 2.
Te development of a drug substance needs to pay attention to its pharmacokinetic properties, as shown in Table 3. HIA shows the percentage of drug absorbed from the cumulative excretion ratio in urine, bile, and feces.Caco-2 is widely used as a model in in vitro testing when predicting human drug absorption through the intestinal epithelial cell barrier [59].Te PPB parameter is related to the ability of the drug disposition to exert an efect [60].At the same time, BBB is used to characterize the distribution of compounds to the blood-brain barrier membrane permeability [59].Cytochrome P450 (CYP450) is an important enzyme system for drug metabolism in the liver, of which CYP2D6 and CYP3A4 are among its subtypes [61].Among CYP enzymes, CYP3A4 and CYP2D6 are the most relevant since they metabolize about 50% and 30% of the drugs on the market, respectively [62].Toxicity in the form of mutagenic and carcinogenic determines the characteristics of compounds causing cell or organ damage when in the body [63].
Quercetin 3-rutinoside has potential antidiarrheal activities based on studies in silico molecular docking.Te presence of quercetin 3-rutinoside in papaya leaves has been proven using ultra performance liquid chromatographytime of fight-electrospray ionization-mass spectrometry methods, together with six other favonoids, namely, quercetin, quercetin 3-(2G-rhamnosylrutinoside), kaempferol, kaempferol 3-rutinoside, kaempferol 3-(2G-rhamnosylrutinoside), and myricetin 3-rhamnoside [64,65].Tis result of molecular docking was also supported by reports on the research results that have been performed on bayberry fruit [61].However, ADMET predictions suggest poor absorption in the gastrointestinal tract and medium permeability in Caco-2 cells.Meanwhile, the distribution has a weak bound so that it can difuse out of the circulatory system and work specifcally on the treatment target [63], and the log BB was not more than 0.3, so it is possible that it is not easy in the blood-brain barrier [66].Prediction of CYP450 showed neither substrate nor inhibitor to CYP2D6, while CYP3A4 inhibitor showed the substrate was weak.Te toxicity test showed that it was neither mutagenic nor carcinogenic; thus, it is safe in the body.

Conclusions
Te phytochemical screening of papaya leaves obtained polyphenols, tannins, favonoids, alkaloids, and saponins.Te antidiarrheal activity of papaya leaves infusion was dosedependent, which the best infusion being 4000 mg/kg.Quercetin 3-rutinoside, a favonoid glycoside, with − 11.50 kcal/mol and 3.75 nM is potentially an antidiarrheal remedy at M 3 -AchR compared as the target to other compounds.However, the prediction of pharmacokinetic properties needs to be considered on several parameters.ADMET prediction showed a specifc distribution to the target and was not toxic, but absorption needed to be considered.

Figure 1 :
Figure 1: Te result was in fve diferent groups (a) total number of feces (mg), (b) onset time of diarrhea (h), and (c) frequency of diarrhea.Group I was negative control, group II was loperamide HCl, group III was infusion 40 mg/mL, group IV was 80 mg/mL, and group V was infusion 160 mg/mL.* Signifcantly diferent when compared with the negative control group at p value <0.05, and values were expressed as mean ± SEM (n � 5).
Swiss Webster male mice aged 2-3 months with 20-25 g of body weight and in a healthy condition were used as experimental animals.Mice were sheltered in polypropylene cages by maintaining laboratory conditions (room temperature 25 ± 2 °C, relative humidity 65-70%; 12 h light/dark cycle) and standard laboratory food and distilled water ad libitum.Te animals were acclimatized to laboratory conditions for 7 days before the experiment.2.4.Ethical Clearance.In vivo experimental procedures and protocols in this study were reviewed and approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Padjadjaran with No. 2142/UN6.KEP/EC/2021.

Table 1 :
Result of the validation method.

Table 3 :
ADMET prediction from the compounds of papaya leaves.
M � mutagenic and C � carcinogenic.